The goal of this proposal is to determine the biochemical mechanisms of regulation of contractile responses to hormones and drugs in heart. Major emphasis will be placed on the role of phosphorylation of specific myofibrillar proteins in altering contractility, and the important biochemical parameters which regulate these phosphorylation reactions in intact tissues. Studies will also be directed towards skeletal muscle to elucidate key differences in the phosphorylation properties of myofibrillar proteins as compared to cardiac muscle. Three experimental approaches will be utilized with studies directed specifically towards phosphorylation of troponin and myosin. First, the physiological state of these muscles in vivo and in selected isolated muscle preparations will be correlated to the phosphorylation of contractile proteins. Pharmacological stimuli as well as ionic perturbations will be used to alter contractile activity in order to determine regulation of the intracellular phosphorylation and dephosphorylation reactions. Measurements will also be made of changes in cyclic nucleotides, protein kinase activities phosphorylase kinase activation and phosphorylase a formation for comparisons to regulation of metabolic events. Secondly, phosphorylation of troponin and myosin by specific kinases will be characterized in vitro with purified proteins. Dephosphorylation of the phosphorylated proteins will also be examined with purified phosphoprotein phosphatases. Thirdly, the biochemical mechanism related to changes in contractile activity will be investigated with the purified proteins. The effect of phosphorylation of troponin and myosin will be evaluated with actomyosin and myosin systems which will allow assessment of both the contractile mechanism itself and calcium regulation of the contractile process. These studies will provide insight into the biochemical mechanisms of regulation of cardiac contractile performance by drugs and hormones.